Project Objective This web page was created as part of an assignment in Emory University's Biology 142 lab course. Students were assigned proteins to be researched, in order to learn about the function and structure of each protein, and determine whether the newly sequenced whale shark genome had evidence of the same proteins. This page is an analysis TICAM1/TRIF.
Background TICAM1/TRIF is a gene that encodes an adaptor protein that plays a role in the immune system. It mediates the recognition of microbes, regulates activation of the innate immune response to bacterial infection, and influences the formation of adaptive immunity. This specific gene is a common polymorphism that modifies the cellular immune response and production of cytokines in vitro (Ferreon et al., n.d.).
TICAM1 specifically encodes for a protein that acts at toll-like receptor 3 (TLR3) during an antiviral immune response (Mish and Hawn 2008). As shown in Figure 1 below, TICAM1 (also known as TRIF) initiate the signaling pathway when double-stranded RNA binds to TLR3. This ligand binding controls the NF-kappa-beta and interferon-regulatory factor (IRF) activation pathways, which in turn induce apoptosis, or cell death, when a pathogen invades (Shirali and Goldstein 2008).
Figure 1. The toll-like receptor (TLR) acts as a system for immune protection. The TLR recruits adaptor proteins, such as TRIF (TICAM1), and initiates signaling pathways in response to the binding of ligands (Shirali).
Methods Whale Shark Orthologs The Galaxy server (whaleshark.georgiaaquarium.org) was utilized to run a BLAST against the predicted whale shark protein database and the human protein sequence (ENSP00000248244) was used as the query. The top five predicted protein hits were identified and the FASTA DNA sequences were extracted. The full predicted whale shark sequences, not just the aligned portion, were used as queries to BLAST against the NCBI human protein database (http://blast.ncbi.nlm.nih.gov).
Predicted Orthologs TRIF predicted orthologs were identified in species other than whale sharks using the NCBI Blast server. The human TRIF protein (ENSP00000248244) was used as query sequence in protein BLASTs against mouse, zebrafish, clawed frog and elephant shark protein databases. Phylogenetic TreeThe human sequence was the query from which the other species' sequences were compare to. The top 5 hits of the whale shark predicted protein along with the TIR domain sequences of the human, zebrafish, elephant shark, mouse, and clawed frog BLAST hits were used to create the phylogenetic tree, specifically using ClustalW2 to create a multiple sequence alignment. ResultsTICAM1/TRIF in the Whale SharkThe results of the BLAST performed in the Galaxy server of the whale shark database with the human protein sequence as the query are shown below in Table 1. Five hits each with an e-value below 1e-01 were identified. The best predicted hit had an e-value of 5e-05.
Whale Shark ID
e-value
Alignment Length
Predicted Protein Length
% Identity
g25712.t1
5e-05
35
712
34.29
g41001.t1
3e-04
39
712
43.59
g34803.t1
3e-04
225
712
24.00
g43334.t1
3e-04
46
712
34.78
g32441.t1
1e-01
71
712
35.21
Table 1. Human TICAM/TRIF top Blastp hits from whale shark predicted protein database. This was done using the Galaxy server from the Georgia Aquarium using the human protein sequence as the query. This table displays whale shark ID, alignment length, and % identity of the top 5 hits according to lowest e-values generated.
The predicted whale shark protein with an ID of g25712.t1 was identified as the best predicted protein. However, the e-value of 5e-05 was larger than expected and the percent identity of 34% was very low. When this sequence and the other four sequences of the top hits was was run against the NCBI human protein database, no orthologs of the TICAMI were identified. However, the TIR domain present in the best hit whale shark sequence was found in the human protein sequence on the NCBI database. The process was repeated to search for TIR domain orthologs in mice, zebrafish, clawed frog, and elephant shark sequences.
Protein domains The best hit TICAM1 proteins in the whale shark (from BLAST results) all contain both a TIR-domain and RHIM-domain. (Figure 2). RHIM domain is involved in virus recognition and is essential for TRIF-induced apoptosis (Rebsamen et al, 2009). TIR domain is necessary for the innate immune response to bacteria and fungi in both Drosophila and mammals.It also plays a key role in activating conserved cellular signal transduction pathways in response to bacterial LPS, microbial and viral pathogens, cytokines and growth factors (Kaiser and Offermann 2005).
TRIF HUMAN BA ELE SHARK.png
Figure 2. Putative domains of the whale shark TICAM1 best hit predicted protein. The best-hit whale shark predicted protein contains putative TIR and RHIM domains as predicted by NCBI BLAST server analyses.
Homologs/Orthologs The human TICAM1 protein sequence (ENSP00000248244) was used as query in NCBI BLAST searches against individual species' protein databases. The best hits for each species were listed in Table 2. TIR domain orthologues were found in mouse, zebrafish, clawed frog and elephant shark, but not necessarily TICAM1 orthologues although TICAM1 protein has been identified in these species.
Species
Name
ID
Length
Query coverage
E-value
Identity
Homo sapiens
TIR domain-containing adapter molecule 1 [Homo sapiens]
NP_891549.1
712
N/A
N/A
N/A
Mouse
TIR domain-containing adapter molecule 1 [Mus musculus]
NP_778154.1
732
99%
0.0
53%
Zebra fish
TIR domain-containing adapter molecule 1 [Danio rerio]
NP_001038224.1
566
53%
2.00E-28
34%
Elephant shark
PREDICTED: TIR domain-containing adapter molecule 1 [Callorhinchus milii]
XP_007899298.1
707
97%
6.00E-54
29%
Clawed frog
PREDICTED: TIR domain-containing adapter molecule 1-like [Xenopus (Silurana) tropicalis]
XP_004911105.1
559
25%
2.00E-33
36%
Table 2. Best hits with human TICAM protein BLAST. The human TICAM1 sequence was used in protein BLASTs against individual species. Name, ID, length, Query coverage, E-value and Identity of the best hit from each search are reported here.
Phylogeny: As shown in figure 2, All five of the best hits from the BLAST search were oldest in relation to the rest of the sequences observed, and were similar to each other. The human and mouse sequences were the youngest, while the zebrafish was closest to the elephant shark, therefore it could be inferred that the zebrafish is a good indicator of the whale shark sequence.
TICAM1.png
Figure 3. The phylogenetic tree was created using the ClustalW2 program to align the top hits of the BLAST searches and other species' sequences based on evolutionary age. Conclusion We were unable to identify a predicted TICAM1/TRIF ortholog in whale sharks. The best predicted whale shark ID g25712.t1 had a high e-value and low query coverage and therefore did not result in a TICAM1 ortholog is any species. However, we were able to identify a TIR domain orthologs across species. The TICAM/TRIF gene encodes a protein in the Toll/interleukin-1 receptor (TIR) homology domain. TIR is an intracellular signaling domain which mediates protein-protein interactions between the toll-like receptors (TLRs) and signal transduction components ("TIR Protein Domain", n.d.). The presence of the TIR domain was used to build a phylogenetic tree which showed that the whale shark ID sequence of g43334.t1 was closest to the clawed frog. This is a surprising result because the g43334.t1 was not the best predicted whale shark protein sequence. Thus, more research needs to be done to investigate the presence and evolution of the TIR domain overtime to identify exactly when the TICAM1 adaptor protein, not present in whale sharks, was lost. This research is important because of TICAM1/TRIF’s significant role in adaptive immunity. References
Kaiser WJ, Offermann MK. “Apoptosis induced by the toll-like receptor adaptor TRIF is dependent on its receptor interacting protein homotypic interaction motif”. J Immunol. 2005 Apr;174(8) 4942-4952. doi:10.4049/jimmunol.174.8.4942. PubMed PMID: 15814722.
Misch, E., & Hawn, T. R.. 1-Toll-like receptor polymorphisms and susceptibility to human disease. Clinical Science (2008). Retrieved April 08, 2015, from http://www.clinsci.org/cs/114/0347/cs1140347.htm
Rebsamen M, Heinz LX, Meylan E, Michallet MC, Schroder K, Hofmann K, Vazquez J, Benedict CA, Tschopp J. “DAI/ZBP1 recruits RIP1 and RIP3 through RIP homotypic interaction motifs to activate NF-kappaB”. EMBO Rep. 10 916-22 200
Shirali, A. C., & Goldstein, D. R. Tracking the Toll of Kidney Disease. Journal of the American Society of Nephrology (2008, August). Retrieved April 08, 2015, from http://jasn.asnjournals.org/content/19/8/1444.full
The predicted whale shark protein with an ID of g25712.t1 was identified as the best predicted protein. However, the e-value of 5e-05 was larger than expected and the percent identity of 34% was very low. When this sequence and the other four sequences of the top hits was was run against the NCBI human protein database, no orthologs of the TICAMI were identified. However, the TIR domain present in the best hit whale shark sequence was found in the human protein sequence on the NCBI database. The process was repeated to search for TIR domain orthologs in mice, zebrafish, clawed frog, and elephant shark sequences.The predicted whale shark protein with an ID of g25712.t1 was identified as the best predicted protein. However, the e-value of 5e-05 was larger than expected and the percent identity of 34% was very low. When this sequence and the other four sequences of the top hits was was run against the NCBI human protein database, no orthologs of the TICAMI were identified. However, the TIR domain present in the best hit whale shark sequence was found in the human protein sequence on the NCBI database. The process was repeated to search for TIR domain orthologs in mice, zebrafish, clawed frog, and elephant shark sequences. The predicted whale shark protein with an ID of g25712.t1 was identified as the best predicted protein. However, the e-value of 5e-05 was larger than expected and the percent identity of 34% was very low. When this sequence and the other four sequences of the top hits was was run against the NCBI human protein database, no orthologs of the TICAMI were identified. However, the TIR domain present in the best hit whale shark sequence was found in the human protein sequence on the NCBI database. The process was repeated to search for TIR domain orthologs in mice, zebrafish, clawed frog, and elephant shark sequences. The predicted whale shark protein with an ID of g25712.t1 was identified as the best predicted protein. However, the e-value of 5e-05 was larger than expected and the percent identity of 34% was very low. When this sequence and the other four sequences of the top hits was was run against the NCBI human protein database, no orthologs of the TICAMI were identified. However, the TIR domain present in the best hit whale shark sequence was found in the human protein sequence on the NCBI database. The process was repeated to search for TIR domain orthologs in mice, zebrafish, clawed frog, and elephant shark sequences.
This web page was created as part of an assignment in Emory University's Biology 142 lab course. Students were assigned proteins to be researched, in order to learn about the function and structure of each protein, and determine whether the newly sequenced whale shark genome had evidence of the same proteins. This page is an analysis TICAM1/TRIF.
Background
TICAM1/TRIF is a gene that encodes an adaptor protein that plays a role in the immune system. It mediates the recognition of microbes, regulates activation of the innate immune response to bacterial infection, and influences the formation of adaptive immunity. This specific gene is a common polymorphism that modifies the cellular immune response and production of cytokines in vitro (Ferreon et al., n.d.).
TICAM1 specifically encodes for a protein that acts at toll-like receptor 3 (TLR3) during an antiviral immune response (Mish and Hawn 2008). As shown in Figure 1 below, TICAM1 (also known as TRIF) initiate the signaling pathway when double-stranded RNA binds to TLR3. This ligand binding controls the NF-kappa-beta and interferon-regulatory factor (IRF) activation pathways, which in turn induce apoptosis, or cell death, when a pathogen invades (Shirali and Goldstein 2008).
Figure 1. The toll-like receptor (TLR) acts as a system for immune protection. The TLR recruits adaptor proteins, such as TRIF (TICAM1), and initiates signaling pathways in response to the binding of ligands (Shirali).
Methods
Whale Shark Orthologs
The Galaxy server (whaleshark.georgiaaquarium.org) was utilized to run a BLAST against the predicted whale shark protein database and the human protein sequence (ENSP00000248244) was used as the query. The top five predicted protein hits were identified and the FASTA DNA sequences were extracted. The full predicted whale shark sequences, not just the aligned portion, were used as queries to BLAST against the NCBI human protein database (http://blast.ncbi.nlm.nih.gov).
Predicted Orthologs
TRIF predicted orthologs were identified in species other than whale sharks using the NCBI Blast server. The human TRIF protein (ENSP00000248244) was used as query sequence in protein BLASTs against mouse, zebrafish, clawed frog and elephant shark protein databases.
Phylogenetic TreeThe human sequence was the query from which the other species' sequences were compare to. The top 5 hits of the whale shark predicted protein along with the TIR domain sequences of the human, zebrafish, elephant shark, mouse, and clawed frog BLAST hits were used to create the phylogenetic tree, specifically using ClustalW2 to create a multiple sequence alignment.
ResultsTICAM1/TRIF in the Whale SharkThe results of the BLAST performed in the Galaxy server of the whale shark database with the human protein sequence as the query are shown below in Table 1. Five hits each with an e-value below 1e-01 were identified. The best predicted hit had an e-value of 5e-05.
The predicted whale shark protein with an ID of g25712.t1 was identified as the best predicted protein. However, the e-value of 5e-05 was larger than expected and the percent identity of 34% was very low. When this sequence and the other four sequences of the top hits was was run against the NCBI human protein database, no orthologs of the TICAMI were identified. However, the TIR domain present in the best hit whale shark sequence was found in the human protein sequence on the NCBI database. The process was repeated to search for TIR domain orthologs in mice, zebrafish, clawed frog, and elephant shark sequences.
Protein domains
The best hit TICAM1 proteins in the whale shark (from BLAST results) all contain both a TIR-domain and RHIM-domain. (Figure 2). RHIM domain is involved in virus recognition and is essential for TRIF-induced apoptosis (Rebsamen et al, 2009). TIR domain is necessary for the innate immune response to bacteria and fungi in both Drosophila and mammals.It also plays a key role in activating conserved cellular signal transduction pathways in response to bacterial LPS, microbial and viral pathogens, cytokines and growth factors (Kaiser and Offermann 2005).
Figure 2. Putative domains of the whale shark TICAM1 best hit predicted protein. The best-hit whale shark predicted protein contains putative TIR and RHIM domains as predicted by NCBI BLAST server analyses.
Homologs/Orthologs
The human TICAM1 protein sequence (ENSP00000248244) was used as query in NCBI BLAST searches against individual species' protein databases. The best hits for each species were listed in Table 2. TIR domain orthologues were found in mouse, zebrafish, clawed frog and elephant shark, but not necessarily TICAM1 orthologues although TICAM1 protein has been identified in these species.
Phylogeny:
As shown in figure 2, All five of the best hits from the BLAST search were oldest in relation to the rest of the sequences observed, and were similar to each other. The human and mouse sequences were the youngest, while the zebrafish was closest to the elephant shark, therefore it could be inferred that the zebrafish is a good indicator of the whale shark sequence.
Figure 3. The phylogenetic tree was created using the ClustalW2 program to align the top hits of the BLAST searches and other species' sequences based on evolutionary age.
Conclusion
We were unable to identify a predicted TICAM1/TRIF ortholog in whale sharks. The best predicted whale shark ID g25712.t1 had a high e-value and low query coverage and therefore did not result in a TICAM1 ortholog is any species. However, we were able to identify a TIR domain orthologs across species. The TICAM/TRIF gene encodes a protein in the Toll/interleukin-1 receptor (TIR) homology domain. TIR is an intracellular signaling domain which mediates protein-protein interactions between the toll-like receptors (TLRs) and signal transduction components ("TIR Protein Domain", n.d.). The presence of the TIR domain was used to build a phylogenetic tree which showed that the whale shark ID sequence of g43334.t1 was closest to the clawed frog. This is a surprising result because the g43334.t1 was not the best predicted whale shark protein sequence. Thus, more research needs to be done to investigate the presence and evolution of the TIR domain overtime to identify exactly when the TICAM1 adaptor protein, not present in whale sharks, was lost. This research is important because of TICAM1/TRIF’s significant role in adaptive immunity.
References
The predicted whale shark protein with an ID of g25712.t1 was identified as the best predicted protein. However, the e-value of 5e-05 was larger than expected and the percent identity of 34% was very low. When this sequence and the other four sequences of the top hits was was run against the NCBI human protein database, no orthologs of the TICAMI were identified. However, the TIR domain present in the best hit whale shark sequence was found in the human protein sequence on the NCBI database. The process was repeated to search for TIR domain orthologs in mice, zebrafish, clawed frog, and elephant shark sequences.The predicted whale shark protein with an ID of g25712.t1 was identified as the best predicted protein. However, the e-value of 5e-05 was larger than expected and the percent identity of 34% was very low. When this sequence and the other four sequences of the top hits was was run against the NCBI human protein database, no orthologs of the TICAMI were identified. However, the TIR domain present in the best hit whale shark sequence was found in the human protein sequence on the NCBI database. The process was repeated to search for TIR domain orthologs in mice, zebrafish, clawed frog, and elephant shark sequences.
The predicted whale shark protein with an ID of g25712.t1 was identified as the best predicted protein. However, the e-value of 5e-05 was larger than expected and the percent identity of 34% was very low. When this sequence and the other four sequences of the top hits was was run against the NCBI human protein database, no orthologs of the TICAMI were identified. However, the TIR domain present in the best hit whale shark sequence was found in the human protein sequence on the NCBI database. The process was repeated to search for TIR domain orthologs in mice, zebrafish, clawed frog, and elephant shark sequences.
The predicted whale shark protein with an ID of g25712.t1 was identified as the best predicted protein. However, the e-value of 5e-05 was larger than expected and the percent identity of 34% was very low. When this sequence and the other four sequences of the top hits was was run against the NCBI human protein database, no orthologs of the TICAMI were identified. However, the TIR domain present in the best hit whale shark sequence was found in the human protein sequence on the NCBI database. The process was repeated to search for TIR domain orthologs in mice, zebrafish, clawed frog, and elephant shark sequences.